DNA immunization is a potentially important approach to vaccination. It has been shown to work in many animal models, producing immune responses which can counter viruses, bacteria, and tumors. However the mechanisms which underpin this approach remain poorly defined. This proposal is aimed towards analyzing these mechanisms, and using the accrued knowledge to manipulate and optimize DNA vaccines. The proposal has four specific aims. Aim 1. To identify the cells which take up and express DNA following intramuscular injection, and which present antigen to T cells. It is known that DNA is expressed in muscle cells; are APCs also transfected? We shall use cloned CTL as probes to identify the cells actually presenting antigen following DNA immunization. Aim 2. To precisely identify which cells induce immunity, and to determine whether muscle cells are important. That cells can be recognized by T cells does not imply that they can induce responses. Can we use cell sorting & transfer to identify the cells responsible for induction of immunity? Aim 3. To evaluate the role of antigen release in DNA immunization, and to identify the underlying mechanisms. Does induction of antibody & CD4+ T cells require antigen release into the humoral phase? If so, what mechanisms underlie this release? Does T cell mediated lysis play a role, and if so, what are the roles of the perforin & fas pathways? Does T cell lysis subsequently limit the immune response? Aim 4. To use the accumulated knowledge to optimize DNA immunization. The knowledge from aims 1-3 will be drawn together to optimize induction of antibodies, CD4+ T cells, and CD8+ T cells each of which probably will have unique requirements for optimal responses.